Impact of the gene polymorphisms in the renin-angiotensin system on cardiomyopathy risk: A meta-analysis

Due to the inconsistent findings from various studies, the role of gene polymorphisms in the renin-angiotensin system in influencing the development of cardiomyopathy remains unclear. In this study, we conducted a systematic review and meta-analysis to summarize the findings regarding the impact of angiotensin converting enzyme (ACE) I/D, angiotensinogen (AGT) M235T, and angiotensin II Type 1 receptor (AGTR1) A1166C gene polymorphisms in patients with cardiomyopathy. We performed a comprehensive search of several electronic databases, including PubMed, Embase, the Cochrane Library, and Web of Science, covering articles published from the time of database creation to April 17, 2023. Studies on the assessment of genetic polymorphisms in genes related to the renin-angiotensin system in relation to cardiomyopathy were included. The primary outcome was cardiomyopathy. Risk of bias was assessed using the Newcastle-Ottawa Scale scale. The meta-analysis includes 19 studies with 4,052 cases and 5,592 controls. The ACE I/D polymorphisms were found to be associated with cardiomyopathy (allelic model D vs I: OR = 1.29, 95CI% = 1.08–1.52; dominant model DD+ID vs II: OR = 1.43, 95CI% = 1.01–2.02; recessive model DD vs ID+II: OR = 0.79, 95CI% = 0.64–0.98). AGT M235T polymorphism and cardiomyopathy were not significantly correlated (allelic model T vs M: OR = 1.26, 95CI% = 0.96–1.66; dominant model TT+MT vs MM: OR = 1.30, 95CI% = 0.98–1.73; recessive model TT vs MT+MM: OR = 0.63, 95CI% = 0.37–1.07). AGTR1 polymorphism and cardiomyopathy were not significantly associated under allelic model A vs C (OR = 0.69, 95CI% = 0.46–1.03) and recessive model AA vs CA+CC (OR = 0.89, 95CI% = 0.34–2.30), but under the dominant model AA+CA vs CC (OR = 0.51, 95CI% = 0.38–0.68). The current meta-analysis reveals that polymorphisms in ACE I/D may be a genetic risk factor for cardiomyopathy. There is an association between AGTR1 gene polymorphisms and risk of cardiomyopathy under the specific model.


Introduction
Cardiomyopathies are heart disorders characterized by structural and functional abnormalities.They include dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), arrhythmogenic right ventricular cardiomyopathy (ARVC), restrictive cardiomyopathy (RCM), and unclassified cardiomyopathy [1].Among these, HCM is the most common genetic heart disease, characterized by left ventricular hypertrophy, which can lead to heart failure and sudden cardiac death.The prevalence of HCM in the general population ranges from 1 in 200 to 1 in 500 for asymptomatic cases and 1 in 3000 for symptomatic cases [2,3].DCM, on the other hand, is a significant genetic heart disease characterized by left ventricular dilation and systolic dysfunction.It is one of the leading causes of heart failure and may require cardiac transplantation in severe cases.The estimated prevalence of DCM in the general population is 1 in 2500 [4].
Over the past three decades, the recognition of disease-causing genetic variants and advancements in understanding the genetic basis of cardiomyopathies have highlighted the importance of genetic factors in their pathogenesis [5][6][7].Among these factors, the reninangiotensin system (RAS) plays a crucial role.The RAS is involved in volume homeostasis and exerts powerful effects on the heart and circulatory systems.In patients with cardiovascular disease, RAS activity is often increased [8].Several reports have indicated that gene polymorphisms in the RAS contribute to the development of cardiomyopathy [9].Key components of the RAS include angiotensin converting enzyme (ACE), angiotensinogen (AGT), and angiotensin II Type 1 receptor (AGTR1).
The ACE gene, located on chromosome 17q23, exhibits a genetic polymorphism in intron 16 characterized by the presence of an insertion (I) or deletion (D) of a non-coding base pair Alu repeat sequence [10].Several studies reported that ACE I/D polymorphism is associated with Cardiomyopathy [11][12][13].AGT, released from the liver, is cleaved by renin.The AGT M235T gene polymorphism, denoting a MET to Thr substitution at codon 235, has been associated with elevated AGT levels.Individuals with the 235TT genotype exhibit 10% to 20% higher plasma AGT levels compared to those with the 235MM genotype [14].The genotype of AGT may serve as a potential risk factor as a positive association has been reported between the AGT M235T polymorphism and HCM [15].The AGTR1 gene, located on chromosomes 3q21-3q25, consists of four introns and five exons, resulting in four distinct alternatively spliced transcripts.More than 20 polymorphisms have been identified in this gene, with the 1166A>C polymorphism in the 3' untranslated region being the most extensively studied in the literature [16].Similarly, linkages between AGTR1 A1166C and cardiomyopathy has been reported [17][18][19].
Despite the studies linking the RAS to cardiomyopathy, the findings from different studies have been contradictory [20][21][22], and the role of the gene polymorphisms in the RAS system remains unclear in influencing the development of cardiomyopathy.Given the inconsistent results from different studies, we conducted a systematic review and meta-analysis to summarize the findings regarding the role of ACE I/D, AGT M235T, and AGTR1 A1166C gene polymorphisms in the RAS in patients with cardiomyopathy.

Materials and methods
This study followed the Preferred Reporting Items for a Systematic Review and Meta-analysis (PRISMA) statement.

Search strategy
We searched PubMed, Embase, the Cochrane Library, and Web of Science for case-control studies assessing the association of genetic polymorphisms in RAS-related genes (ACE I/D, AGT M235T, and AGTR1 A1166C) with cardiomyopathy, with a time limit of from database creation to April 17, 2023.Cardiomyopathy, RAS-associated genes, gene polymorphisms, and case-controls were searched individually or in combination, and English was the only language allowed.Only human studies were included, and the search strategy is detailed in the S1 Table.

Study selection
Inclusion criteria were as follows: (1) studies that included patients with cardiomyopathy; (2) studies that investigated any gene of ACE I/D, AGT M235T, or AGTR1 A1166C; (3) study design should be a case-control study; and (4) studies that were published in English.Exclusion criteria were (1) abstracts, letters, conference talks, or reviews; (2) studies that involved patients with non-cardiomyopathy; (3) duplicate studies that provided overlapping data; (4) studies that did not provide sufficient data for meta-analysis.Two independent reviewers completed the study selection with disagreement resolved by consensus.

Data extraction
Three reviewers independently reviewed all included studies and extracted the following data: author, year of publication, region of the dataset, ethnicity of the study population, type of cardiomyopathy, genotype, genotyping method, sample size of the case and control groups, and whether the control group was validated with Hardy-Weinberg principle.Two independent reviewers completed the data extraction with disagreement resolved by consensus.

Quality assessment
Three reviewers independently rated the quality of the included case-control studies using the Newcastle-Ottawa Scale (NOS).It evaluates studies by means of three major blocks of eight items, specifically study population selection (selection), comparability (comparability), and exposure (exposure) evaluations.The NOS evaluates the quality of the literature using the semi-quantitative principle of the star system, with a total score of 9 stars [23,24].Two independent reviewers completed the quality assessment with disagreement resolved by consensus.

Statistical methods
Data were analyzed using STATA version 15.1.The Cochran Q test and I 2 statistics were employed to analyze study heterogeneity, and I 2 was calculated using the equation: I 2 = 100% (Q df)/Q.I-square less than 50% was considered as low heterogeneity between studies and greater than 50% was considered as high heterogeneity [25,26].Sources of heterogeneity were explored by subgroup analysis.The significance of the combined odds ratio (OR) was determined by the Z-test, in which P � 0.05 was considered significant.
The main characteristics of the included studies are shown in Table 1.The 19 studies encompass 4,052 patients with cardiomyopathy.Among these participants, 1,554 individuals were diagnosed with HCM, while 2,498 individuals were diagnosed with DCM.Additionally, we included a control group consisting of 5,592 individuals.In all of the 19 studies, echocardiography was used as the diagnostic tool for cardiomyopathy.The included studies were published between 1995 and 2019, covering a wide time range of research in this field.Regarding the method of genetic testing, 17 studies [20,27,28,30,31,[33][34][35][36][37][38][39][40][41][42][43][44] employed the polymerase chain reaction (PCR) technique, while methods were unclear in two studies [29,32].

Association of ACE I/D genes polymorphism with the risk of cardiomyopathy
Twenty studies explored the association between ACE I/D gene polymorphism and the risk of cardiomyopathy.ACE I/D polymorphisms were found to be associated with cardiomyopathy  A subgroup analysis was conducted based on the type of cardiomyopathy (HCM and DCM) and whether the control group used Hardy-Weinberg validation (yes and no).The result is presented in Table 2. Regarding the type of cardiomyopathy, an association was observed between ACE I/D variants and HCM risk under the dominant model, with a pooled OR of 1.820 (DD + ID vs. II: 95% CI = 1.040-3.183;P = 0.036).Additionally, associations were detected between ACE I/D variants and DCM risk under the allelic and recessive models, with pooled ORs of 1.287 (D vs I: 95% CI = 1.043-1.588;P = 0.019), 0.746 (DD vs ID + II: 95% CI = 0.577-0.965;P = 0.025), respectively.In studies validated with Hardy-Weinberg principle, there was an association between ACE I/D variants and cardiomyopathy risk under the recessive model, with a pooled OR of 0.779 (DD vs ID+II: 95% CI = 0.607-1.000;P = 0.050).In studies validated without Hardy-Weinberg principle, there was an association between ACE I/ D variants and risk of cardiomyopathy with pooled ORs of 1.647 under the allelic model (D vs I: 95% CI = 1.138-2.384;P = 0.008) and 2.433 under dominant model (DD + ID vs II: 95%   CI = 1.151-5.142;P = 0.020).Heterogeneity between studies across subgroups was not eliminated.In the subgroup analysis, an association was detected between the AGT M235T polymorphism and DCM risk under the dominant model with a pooled OR of 2.090 (TT+MT vs MM: 95% CI = 1.162-3.759;P = 0.014).No association was found between the remaining subgroups of AGT M235T (Table 2).Inter-study heterogeneity was eliminated in the HCM, DCM, and NHW groups (TT+MT vs MM: P heterogeneity = 0.547, I2 = 0.0%; TT+MT vs MM: P heterogene- ity = 0.459, I2 = 0.0%; TT+MT vs MM: P heterogeneity = 0.175, I2 = 39.5%).

Association of AGTR1 A1166C genes polymorphism with the risk of cardiomyopathy
Among the four studies included in our meta-analysis that investigated the association between AGTR1 A1166C polymorphisms and the risk of cardiomyopathy, no significant association was observed when analyzing the allelic model A vs C (OR = 0.69, 95% CI = 0.The results of subgroup analysis are shown in Table 2.An association between AGTR1 A1166C polymorphism and HCM risk was detected under the dominant model with a pooled OR of 0.523 (AA+CA vs CC: 95% CI = 0.363-0.753;P = 0.001).Associations were detected between AGTR1 polymorphisms and DCM risk under the allelic model and dominant model, with pooled ORs of 0.548 (A vs C: 95% CI = 0.362-0.828;P = 0.004) and 0.488 (AA+CA vs CC: 95% CI = 0.311-0.766;P = 0.002), respectively.In studies validated with Hardy-Weinberg principle, there was an association between AGTR1 polymorphism and cardiomyopathy risk under the dominant model with a pooled OR of 4.289 (AA+CA vs CC: 95% CI = 0.219-83.934;P = 0.001).In studies validated without Hardy-Weinberg principle, there was an association between AGTR1 polymorphism and risk of cardiomyopathy with pooled ORs of 0.595 (allelic model, A vs C: 95% CI = 0.451-0.785;P = 0.008) and 0.492 (dominant model, AA+CA vs CC: 95% CI = 0.369-0.1655;P = 0.001).Heterogeneity between studies was not eliminated except in the NHW group (A vs C: P heterogeneity = 0.191, I 2 = 39.7%;AA+CA vs CC: P heterogeneity = 0.184, I 2 = 41.0%).

Quality assessment
Newcastle-Ottawa Scale (NOS) scores for the included studies are shown in S2 Table .All studies included consecutive or representative series of cases, and all studies controlled for the most important confounding factors.Only a small number of studies did not describe the control group in question.Twenty-nine studies were of good quality, with a quality assessment score of 7 or more (out of 9).

Publication bias
The funnel plots for all the above studies are presented in S1 Fig.No publication bias was observed, as the shape of the funnel plots did not seem to show significant asymmetry in each meta-analysis.This was further verified by Egger's test (ACE D/I: P = 0.149, AGT M235T: P = 0.149, AGTR1 A1166C: P = 0.349).

Discussion
In this meta-analysis, we reviewed all eligible studies that evaluate the associations between ACE I/D, AGT M235T, and AGTR1 A1166C gene polymorphisms and the risk of cardiomyopathy.The ACE I/D gene polymorphisms were found to be associated with cardiomyopathy, while M235T and AGTR1 A1166C gene polymorphisms and cardiomyopathy were not significantly correlated.
ACE plays a crucial role in the synthesis of angiotensin II (Ang II), which in turn triggers various cellular processes such as proliferation, migration, hypertrophy, as well as the upregulation of proinflammatory cytokines and matrix metalloproteinases.Consequently, the overexpression of Ang II has been implicated in the development of cardiomyopathy.In the present study, ACE I/D gene polymorphism was found to be associated with an increased risk of DCM and HCM, which is consistent with previous meta-analysis.Yuan et al. conducted a meta-analysis comprising 2,972 participates and found that D allele carrier is a risk allele, which indicates that ACE gene polymorphism may be a genetic risk factor for HCM [13].Luo conducted a meta-analysis comparing the DI/II genotype to DD genotype and found an OR of 0.73, which indicated the potential association between ACE I/D gene polymorphism and HCM [57].In a meta-analysis conducted by Shen et al. [58], ACE I/D gene polymorphism was associated with the increased risk of DCM/HCM in Asians, but not in Caucasians.In a meta-analysis conducted by Yang, ACE I/D polymorphism may be associated with HCM but not DCM susceptibility [59].
Extensive attention has been devoted to investigating the potential link between the AGT M235T polymorphism and the development of cardiomyopathy.Previous studies have yielded divergent outcomes.Kawaguchi's study identified the gene polymorphism of M235T as a predisposing factor for cardiac hypertrophy in HCM [42].Likewise, Ranni et al. [55] reported an association between the M235T polymorphism and DCM, HCM and RCM.In contrast, Tiago et al. [46] observed no correlation between the M235T polymorphism and DCM in Black South Africans.Tiret et al. [48] explored a cohort of 428 participants with DCM and found no link between the M235T polymorphism and either the risk or severity of DCM.Similarly, Coto et al. [50] did not identify an association between M235T and HCM.Upon pooling the results, our analysis revealed no significant correlation between AGT M235T polymorphism and cardiomyopathy.However, subgroup analysis identified that the AGT M235T polymorphism is association with increased DCM risk in the dominant model, which indicates that the presence of one or two copies of T allele significantly increases the risk of DCM.It has been reported that elevated levels of serum AGT were observed in the heart and kidney of individuals who possess the 235T allele [60].AGT plays a pivotal role in the production of angiotensin II through its interaction with renin [61].Angiotensin II, in turn, has been demonstrated to promote vasoconstriction, increases blood pressure, and stimulates the release of aldosterone that may ultimately lead to the development of DCM.While the AGT M235T gene polymorphism is associated with an increased DCM risk, it remains unclear whether this genetic variant directly contributes to the pathogenesis of DCM or if it serves as a marker for other genetic or environmental factors.Additional research, including functional studies and animal models, is needed to establish a causal link and to uncover the precise mechanisms by which this gene polymorphism influences DCM susceptibility.
A noteworthy observation was made regarding the increased frequency of AGTR1 A1166C carriers among the HCM patients without sarcomeric mutations, implying a potential risk associated with the AGTR1 A1166C polymorphism in HCM [50].Similarly, Rani's study reported a higher prevalence of AGTR1 1166CC in both HCM and DCM cases [55].However, a study conducted in a Japanese cohort failed to establish an association between AGTR1 A1166C gene polymorphisms and HCM [56].Our comprehensive analysis, encompassing all available data, did not reveal a significant correlation between the AGTR1 A1166C polymorphism and cardiomyopathy.Nevertheless, our findings support an association between the AGTR1 A1166C gene polymorphism and HCM risk under the dominant model, as well as an association with DCM under the allelic and dominant models.Previous meta-analyses confirmed the association of the AGTR1 A1166C polymorphism with coronary heart disease [62,63] and essential hypertension [64].However, only limited studies have investigated its relationship with cardiomyopathy.To the best of our knowledge, this is the first meta-analysis examining the association between AGTR1 A1166C and cardiomyopathy.Further high-quality and large-scale trials are warranted to shed more light on this topic.
It is important to interpret the results of our meta-analysis with caution due to several limitations.Firstly, heterogeneity is a common issue encountered in meta-analyses of association studies and it was also observed in our study, which could influence the results.The heterogeneity may arise from several factors, including variations in study designs, or differences in sample selection among the included studies.Secondly, previous literature has reported that the association between ACE D/I polymorphism and M235T polymorphism with cardiomyopathy may vary among different ethnicities.However, we were unable to perform subgroup analyses based on different ethnicities due to the limited number of studies available for each specific ethnicity.Thirdly, gene-gene interactions may also contribute to the development of cardiomyopathy.However, limited information was included in the original studies.These limitations highlight the need for further research to explore the potential impact of different ethnicities and gene-environment interactions on the association between ACE D/I, AGTR1 A1166C, and M235T polymorphisms with cardiomyopathy.

Conclusion
The current meta-analysis suggests that polymorphisms in the ACE I/D may be a genetic risk factor for cardiomyopathy.The dominant model detected associations between ACE I/D polymorphisms and HCM risk, whereas the allelic and recessive models detected associations between ACE I/D polymorphisms and DCM risk.Under the dominant hypothesis, an association between AGT M235T polymorphism and DCM risk was discovered.Furthermore, a relationship was discovered between AGTR1 polymorphism and HCM risk under the dominant model, as well as an association between AGTR1 polymorphism and DCM risk under the allelic model and the dominant model.

Five
studies have been conducted to investigate the relationship between AGT M235T polymorphism and the risk of cardiomyopathy.AGT M235T polymorphism and cardiomyopathy were not significantly correlated.Under the allelic model T vs M, a pooled OR of 1.26 (95 CI% = 0.96-1.66) is observed (Fig 3A).Under the dominant model TT+MT vs MM, the pooled OR is 1.30 (95 CI% = 0.98-1.73,Fig 3B), while under the recessive model TT vs MT+MM, the pooled OR is 0.63 (95 CI% = 0.37-1.07,Fig 3C).